Rapamycin amidino carbamates

ABSTRACT

A compound of the structure ##STR1## wherein R and R 1  are each, independently, hydrogen, or ##STR2## R 2  and R 3  are each, independently, hydrogen, alkyl, alkenyl, alkynyl, --CO 2  R 5 , --COR 5 , --CN, --NO 2 , --SO 2  R 5 , --SO 3  R 5 , --OR 5 , --SR 5 , or Ar; 
     R 4  is hydrogen, alkyl, alkenyl, alkynyl, --CF 3 , --NR 5  R 6 , --CO 2  R 5 , --COR 5 , CONR 5  R 6 , --NO 2 , halogen, --OR 5 , --SR 5 , --CN, --SO 2  R 5 , --SO 3  R 5 , --SO 2  NR 5  R 6 , or Ar; 
     R 5  and R 6  are each, independently, hydrogen, alkyl, alkenyl, alkynyl, or Ar; 
     Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may be optionally substituted; with the proviso that R and R 1  are both not hydrogen, or a pharmaceutically acceptable salt thereof which is useful as an immunosuppressive, antiinflammatory, antifungal, antiproliferative, and antitumor agent.

This is a division of application Ser. No. 08/259,763 filed Jun. 14,1994, now U.S. Pat. No. 5,463,048.

BACKGROUND OF THE INVENTION

This invention relates to amidino carbamates of rapamycin and a methodfor using them for inducing immunosuppression, and in the treatment oftransplantation rejection, graft vs. host disease, autoimmune diseases,diseases of inflammation, adult T-cell leukemia/lymphoma, solid tumors,fungal infections, and hyperproliferative vascular disorders.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiot. 28, 727(1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Pat. No.3,929,992; and U.S. Pat. No. 3,993,749].

Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)]disclosed that rapamycin is effective in the experimental allergicencephalomyelitis model, a model for multiple sclerosis; in the adjuvantarthritis model, a model for rheumatoid arthritis; and effectivelyinhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411(1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978);and U.S. Pat. No. 5,100,899].

Rapamycin has also been shown to be useful in preventing or treatingsystemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonaryinflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetesmellitus [Fifth Int. Conf. Inflamm. Res. Assoc. 121 (Abstract), (1990)],smooth muscle cell proliferation and intimal thickening followingvascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197(1992)], adult T-cell leukemia/lymphoma [European Patent Application525,960 A1], and ocular inflammation [European Patent Application532,862 A1].

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and43 positions) have been shown to be useful as antifungal agents (U.S.Pat. No. 4,316,885) and used to make water soluble aminoacyl prodrugs ofrapamycin (U.S. Pat. No. 4,650,803). Recently, the numbering conventionfor rapamycin has been changed; therefore according to ChemicalAbstracts nomenclature, the esters described above would be at the 31-and 42- positions. U.S. Pat. Nos. 5,118,678 and 5,302,584 disclosescarbamates of rapamycin that are useful as immunosuppressive,antiinflammatory, antifungal, antiproliferative, and antitumor agents.

DESCRIPTION OF THE INVENTION

This invention provides derivatives of rapamycin which are useful asimmunosuppressive, antiinflammatory, antifungal, antiproliferative, andantitumor agents having the structure ##STR3## wherein R and R¹ areeach, independently, hydrogen, or ##STR4## R² and R³ are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7carbon atoms, alkynyl of 2-7 carbon atoms, --CO₂ R⁵, --COR⁵, --CN,--NO₂, --SO₂ R⁵, --SO₃ R⁵, --OR⁵, --SR⁵, or Ar;

R⁴ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, --CF₃, --NR⁵ R⁶, --CO₂ R⁵, --COR⁵, CONR⁵R⁶, --NO₂, halogen, --OR⁵, --SR⁵, --CN, --SO₂ R⁵, --SO₃ R⁵, --SO₂ NR⁵R⁶, or Ar;

R⁵ and R⁶ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or Ar;

Ar is phenyl, naphthyl, or hetaryl, wherein the foregoing may beoptionally mono-, di-, or tri-substituted with a group selected fromalkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbonatoms, cyano, halo, hydroxy, nitro, carbalkoxy of 2-7 carbon atoms,trifluoromethyl, trifluoromethoxy, amino, dialkylamino of 1-6 carbonatoms per alkyl group, dialkylaminoalkyl of 3-12 carbon atoms,hydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms,alkylthio of 1-6 carbon atoms, --SO₃ H, and --CO₂ H;

with the proviso that R and R¹ are both not hydrogen, or apharmaceutically acceptable salt thereof.

The pharmaceutically acceptable salts are those derived from suchinorganic cations such as sodium, potassium, and the like; organic basessuch as: mono-, di-, and trialkyl amines of 1-6 carbon atoms, per alkylgroup and mono-, di-, and trihydroxyalkyl amines of 1-6 carbon atoms peralkyl group, and the like; and organic and inorganic acids as: acetic,lactic, citric, tartaric, succinic, maleic, malonic, gluconic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, and similarly known acceptable acids.

The terms alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, andalkynyl of 2-7 carbon atoms, include both straight chain as well asbranched carbon chains. When any of the generic terms (i.e., R⁵) arecontained more than once in a given compound, each may be the same ordifferent.

Hetaryl is defined as an unsaturated or partially saturated heterocyclicradical of 5-12 atoms having 1 ring or 2 fused rings. Preferredheterocyclic radicals include unsaturated heterocyclic radicals such asfuranyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2-dithiolyl, 1,3-dithiolyl, 1,2,3-oxathiolyl,isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, 1,2,3-oxadiazolyl,1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl,1,2,3,5-oxatriazolyl, 1,2,3-dioxazolyl, 1,2,4-dioxazolyl,1,3,2-dioxazolyl, 1,3,4-dioxazolyl, 1,2,5-oxathiazolyl, 1,3-oxathiolyl,1,2-pyranyl, 1,4-pyranyl, pyridinyl, pyridazinyl, pyrimidinyl,pyrazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl,1,2,4-oxazinyl, 1,3,2-oxazinyl, 1,2,6-oxazinyl, 1,4-oxazinyl,isoxazinyl, 1,2,5-oxathiazinyl, 1,4-oxazinyl, o-isoxazinyl,p-isoxazinyl, 1,2,5-oxathiazinyl, 1,2,6-oxathiazinyl,1,3,5,2-oxadiazinyl, azepinyl, oxepinyl, thiepinyl, 1,2,4-diazepinyl,benzofuranyl, isobenzofuranyl, thionaphthenyl, indolyl, indolenyl,2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, benzpyrazolyl,benzisoxazolyl, benzoxazolyl, anthranilyl, 1,2-benzopyranyl, quinolinyl,isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl,pyrido[3,4-b]pyridinyl, pyrido[4,3-b]pyridinyl, pyrido[2,3-b]pyridinyl,1,3,2-benzozazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl,3,1,4-benzoxazinyl, 1,2-benzisoxazinyl, 1,4-benzisoxazinyl, carbazolyl,purinyl, and partially saturated heterocyclic radicals selected from thelist above. All of the preferred heterocyclic radicals contain at leastone double bond. When the heterocyclic radical is partially saturated,one or more of the olefins in the unsaturated ring system is saturated;the partially saturated heterocyclic radical still contains at least onedouble bond. It is more preferred that hetaryl is pyridinyl.

Of the compounds of this invention preferred members are those in whichR¹ is hydrogen.

Compounds of this invention having the amidino carbamate moiety at the42- or 31,42-positions can be prepared by conversion of rapamycin to the4-nitrophenylcarbonate (as illustrated in Example 1), followed byreaction with an appropriately functionalized amidine. Mixtures of 42-and 31,42-carbamates can be separated by chromatography.

The 31-amidino carbamates of this invention can be prepared byprotecting the 42-alcohol of rapamycin with a protecting group, such aswith a tert-butyl dimethylsilyl group, followed by carbamylation of the,31-position by the procedures described above. The preparation ofrapamycin 42-silyl ethers is described in U.S. Pat. No. B1 5,120,842,which is hereby incorporated by reference. Removal of the protectinggroup provides the 31-esterified compounds. In the case of thetert-butyl dimethylsilyl protecting group, deprotection can beaccomplished under mildly acidic conditions, such as aceticacid/water/THF. The deprotection procedure is described in Example 15 ofU.S. Pat. No. 5,118,678, which is hereby incorporated by reference.

Having the 31-position carbamylated and the 42-position deprotected, the42-position can be carbamylated using a different amidine agent than wasreacted with the 31-carbonate, to give compounds having differentamidino carbamates at the 31- and 42- positions. Alternatively, the42-carbamylated compounds, prepared as described above, can be convertedto the 31-carbonate-42-carbamate and reacted with a different amidine toprovide compounds having different carbamates at the 31- and42-positions.

This invention also covers analogous carbamates of other rapamycins suchas, but not limited to, 29-demethoxyrapamycin, [U.S. Pat. No. 4,375,464,32-demethoxyrapamycin under C.A. nomenclature]; rapamycin derivatives inwhich the double bonds in the 1-, 3-, and/or 5-positions have beenreduced [U.S. Pat. No. 5,023,262]; 29-desmethylrapamycin [U.S. Pat. No.5,093,339, 32-desmethylrapamycin under C.A. nomenclature];7,29-bisdesmethylrapamycin [U.S. Pat. No. 5,093,338,7,32-desmethylrapamycin under C.A. nomenclature]; and15-hydroxyrapamycin [U.S. Pat. No. 5,102,876]. The disclosures in theabove cited U.S. Patents are hereby incorporated by reference.

Immunosuppressive activity for representative compounds of thisinvention was evaluated in an in vitro standard pharmacological testprocedure to measure the inhibition of lymphocyte proliferation (LAF)and in two in vivo standard pharmacological test procedures. The pinchskin graft test procedure measures the immunosuppressive activity of thecompound tested as well as the ability of the compound tested to inhibitor treat transplant rejection. The adjuvant arthritis standardpharmacological test procedure, which measures the ability of thecompound tested to inhibit immune mediated inflammation. The adjuvantarthritis test procedure is a standard pharmacological test procedurefor rheumatoid arthritis. The procedures for these standardpharmacological test procedures are provided below.

The comitogen-induced thymocyte proliferation procedure (LAF) was usedas an in vitro measure of the immunosuppressive effects ofrepresentative compounds. Briefly, cells from the thymus of normalBALB/c mice are cultured for 72 hours with PHA and IL-1 and pulsed withtritiated thymidine during the last six hours. Cells are cultured withand without various concentrations of rapamycin, cyclosporin A, or testcompound. Cells are harvested and incorporated radioactivity isdetermined. Inhibition of lymphoproliferation is assessed as percentchange in counts per minute from non-drug treated controls. For eachcompound evaluated, rapamycin was also evaluated for the purpose ofcomparison. An IC₅₀ was obtained for each test compound as well as forrapamycin. When evaluated as a comparator for the representativecompounds of this invention, rapamycin had an IC₅₀ ranging from 0.4-1.0nM. The results obtained are provided as an IC₅₀ and as the percentinhibition of T-cell proliferation at 0.1 μM. The results obtained forthe representative compounds of this invention were also expressed as aratio compared with rapamycin. A positive ratio indicatesimmunosuppressive activity. A ratio of greater than 1 indicates that thetest compound inhibited thymocyte proliferation to a greater extent thanrapamycin. Calculation of the ratio is shown below. ##EQU1##

Representative compounds of this invention were also evaluated in an invivo test procedure designed to determine the survival time of pinchskin graft from male BALB/c donors transplanted to male C₃ H(H-2K)recipients. The method is adapted from Billingham R. E. and Medawar P.B., J. Exp. Biol. 28:385-402, (1951). Briefly, a pinch skin graft fromthe donor was grafted on the dorsum of the recipient as a allograft, andan isograft was used as control in the same region. The recipients weretreated with either varying concentrations of test compoundsintraperitoneally or orally. Rapamycin was used as a test control.Untreated recipients serve as rejection control. The graft was monitoreddaily and observations were recorded until the graft became dry andformed a blackened scab. This was considered as the rejection day. Themean graft survival time (number of days ±S.D.) of the drug treatmentgroup was compared with the control group. The following table shows theresults that were obtained. Results are expressed as the mean survivaltime in days. Untreated (control) pinch skin grafts are usually rejectedwithin 6-7 days. Compounds were tested using a dose of 4 mg/kg.

The adjuvant arthritis standard pharmacological test procedure measuresthe ability of test compounds to prevent immune mediated inflammationand inhibit or treat rheumatoid arthritis. The following brieflydescribes the test procedure used. A group of rats (male inbread WistarLewis rats) are pre-treated with the compound to be tested (1 h prior toantigen) and then injected with Freud's Complete Adjuvant (FCA) in theright hind paw to induce arthritis. The rats are then orally dosed on aMonday, Wednesday, Friday schedule from day 0-14 for a total of 7 doses.Both hind paws are measured on days 16, 23, and 30. The difference inpaw volume (mL) from day 16 to day 0 is determined and a percent changefrom control is obtained. The left hind paw (uninjected paw)inflammation is caused by T-cell mediated inflammation and is recordedin the above table (% change from control). The right hind pawinflammation, on the other hand, is caused by nonspecific inflammation.Compounds were tested at a dose of 5 mg/kg. The results are expressed asthe percent change in the uninjected paw at day 16 versus control; themore negative the percent change, the more potent the compound.Rapamycin provided -90% change versus control, indicating that rapamycintreated rats had 90% less immune induced inflammation than control rats.

The results obtained in these standard pharmacological test proceduresare provided following the procedure for making the specific compoundsthat were tested.

The results of these standard pharmacological test proceduresdemonstrate immunosuppressive activity both in vitro and in vivo for thecompounds of this invention. The results obtained in the LAF testprocedure indicates suppression of T-cell proliferation, therebydemonstrating the immunosuppressive activity of the compounds of thisinvention. Further demonstration of the utility of the compounds of thisinvention as immunosuppressive agents was shown by the results obtainedin the skin graft and adjuvant arthritis standard pharmacological testprocedures. Additionally, the results obtained in the skin graft testprocedure further demonstrates the ability of the compounds of thisinvention to treat or inhibit transplantation rejection. The resultsobtained in the adjuvant arthritis standard pharmacological testprocedure further demonstrate the ability of the compounds of thisinvention to treat or inhibit rheumatoid arthritis.

Based on the results of these standard pharmacological test procedures,the compounds are useful in the treatment or inhibition oftransplantation rejection such as kidney, heart, liver, lung, bonemarrow, pancreas (islet cells), cornea, small bowel, and skinallografts, and heart valve xenografts; in the treatment or inhibitionof graft vs. host disease; in the treatment or inhibition of autoimmunediseases such as lupus, rheumatoid arthritis, diabetes mellitus,myasthenia gravis, and multiple sclerosis; and diseases of inflammationsuch as psoriasis, dermatitis, eczema, seborrhea, inflammatory boweldisease, pulmonary inflammation (including asthma, chronic obstructivepulmonary disease, emphysema, acute respiratory distress syndrome,bronchitis, and the like), and eye uveitis.

Because of the activity profile obtained, the compounds of thisinvention also are considered to have antitumor, antifungal activities,and antiproliferative activities. The compounds of this inventiontherefore also useful in treating solid tumors, adult T-cellleukemia/lymphoma, fungal infections, and hyperproliferative vasculardiseases such as restenosis and atherosclerosis. When used forrestenosis, it is preferred that the compounds of this invention areused to treat restenosis that occurs following an angioplasty procedure.When used for this purpose, the compounds of this invention can beadministered prior to the procedure, during the procedure, subsequent tothe procedure, or any combination of the above.

When administered for the treatment or inhibition of the above diseasestates, the compounds of this invention can be administered to a mammalorally, parenterally, intranasally, intrabronchially, transdermally,topically, intravaginally, or rectally.

It is contemplated that when the compounds of this invention are used asan immunosuppressive or antiinflammatory agent, they can be administeredin conjunction with one or more other immunoregulatory agents. Suchother immunoregulatory agents include, but are not limited toazathioprine, corticosteroids, such as prednisone andmethylprednisolone, cyclophosphamide, rapamycin, cyclosporin A, FK-506,OKT-3, and ATG. By combining the compounds of this invention with suchother drugs or agents for inducing immunosuppression or treatinginflammatory conditions, the lesser amounts of each of the agents arerequired to achieve the desired effect. The basis for such combinationtherapy was established by Stepkowski whose results showed that the useof a combination of rapamycin and cyclosporin A at subtherapeutic dosessignificantly prolonged heart allograft survival time. [TransplantationProc. 23: 507 (1991)].

The compounds of this invention can be formulated neat or with apharmaceutical carrier to a mammal in need thereof. The pharmaceuticalcarrier may be solid or liquid. When formulated orally, it has beenfound that 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentrate with1,2-propylene glycol, A. Nattermann & Cie. GmbH) provides an acceptableoral formulation.

A solid carrier can include one or more substances which may also act asflavoring agents, lubricants, solubilizers, suspending agents, fillers,glidants, compression aids, binders or tablet-disintegrating agents; itcan also be an encapsulating material. In powders, the carrier is afinely divided solid which is in admixture with the finely dividedactive ingredient. In tablets, the active ingredient is mixed with acarrier having the necessary compression properties in suitableproportions and compacted in the shape and size desired. The powders andtablets preferably contain up to 99% of the active ingredient. Suitablesolid carriers include, for example, calcium phosphate, magnesiumstearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose,methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine,low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions,syrups, elixirs and pressurized compositions. The active ingredient canbe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid carrier can containother suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, colors, viscosity regulators,stabilizers or osmo-regulators. Suitable examples of liquid carriers fororal and parenteral administration include water (partially containingadditives as above, e.g. cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, e.g. glycols) and their derivatives,lethicins, and oils (e.g. fractionated coconut oil and arachis oil). Forparenteral administration, the carrier can also be an oily ester such asethyl oleate and isopropyl myristate. Sterile liquid carriers are usefulin sterile liquid form compositions for parenteral administration. Theliquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions which am sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

The compounds of this invention may be administered rectally in the formof a conventional suppository. For administration by intranasal orintrabronchial inhalation or insufflation, the compounds of thisinvention may be formulated into an aqueous or partially aqueoussolution, which can then be utilized in the form of an aerosol. Thecompounds of this invention may also be administered transdermallythrough the use of a transdermal patch containing the active compoundand a carrier that is inert to the active compound, is non toxic to theskin, and allows delivery of the agent for systemic absorption into theblood sire am via the skin. The carrier may take any number of formssuch as creams and ointments, pastes, gels, and occlusive devices. Thecreams and ointments may be viscous liquid or semisolid emulsions ofeither the oil-in-water or water-in-oil type. Pastes comprised ofabsorptive powders dispersed in petroleum or hydrophilic petroleumcontaining the active ingredient may also be suitable. A variety ofocclusive devices may be used to release the active ingredient into theblood stream such as a semipermiable membrane covering a reservoircontaining the active ingredient with or without a carrier, or a matrixcontaining the active ingredient. Other occlusive devices are known inthe literature.

In addition, the compounds of this invention may be employed as asolution, cream, or lotion by formulation with pharmaceuticallyacceptable vehicles containing 0.1-5 percent, preferably 2%, of activecompound which may be administered to a fungally affected area.

The dosage requirements vary with the particular compositions employed,the route of administration, the severity of the symptoms presented andthe particular subject being treated. Based on the results obtained inthe standard pharmacological test procedures, projected dally dosages ofactive compound would be 0.1 μg/kg-100 mg/kg, preferably between0.001-25 mg/kg, and more preferably between 0.01-5 mg/kg. Treatment willgenerally be initiated with small dosages less than the optimum dose ofthe compound. Thereafter the dosage is increased until the optimumeffect under the circumstances is reached; precise dosages for oral,parenteral, nasal, or intrabronchial administration will be determinedby the administering physician based on experience with the individualsubject treated. Preferably, the pharmaceutical composition is in unitdosage form, e.g. as tablets or capsules. In such form, the compositionis sub-divided in unit dose containing appropriate quantities of theactive ingredient; the unit dosage forms can be packaged compositions,for example, packeted powders, vials, ampoules, prefilled syringes orsachets containing liquids. The unit dosage form can be, for example, acapsule or tablet itself, or it can be the appropriate number of anysuch compositions in package form.

The following examples illustrate the preparation and biologicalactivities of representative compounds of this invention.

EXAMPLE 1 42-O-(4-Nitro-phenoxycarbonyl)rapmycin

To a solution of 5.15 g (5.633 mmol) of rapamycin in 40 ml of methylenechloride cooled to -78° C. with dry ice/acetone bath, was added 0.7 mldry pyridine and 1.70 g (8.450 mmol) of p-nitrophenylchloroformatedissolved in 10 ml methylene chloride. The reaction mixture was allowedto warm to ambient and stirred overnight under nitrogen. The reactionmixture was concentrated in vacuo and partitioned between ether andwater. The organic phase was washed with 0.1N HCl (3×), then with asaturated sodium chloride solution (2×), dried over magnesium sulfate,filtered and concentrated under vacuum to give a pale yellow solid.Purification by flash column chromatography (elution with 40% then 50%ethyl acetate/hexanes) gave 5.41 g (88%) of the title compound as a paleyellow solid.

¹ H NMR (DMSO) δ 8.3 and 7.5 (d and d, aromatic-H, 4H), 4.5 (m, 42C-H,1H).

MS (-) FAB m/z: 1078 (M⁻), 590 (Southern Fragment).

EXAMPLE 2 Rapamycin 42-ester with (imino-phenyl-methyl)carbamic acid

To a solution of 1.0024 g (0.9287 mmol) of42-O-(4-Nitrophenoxycarbonyl)rapamycin in 5 ml of DMF was added 0.2231 g(1.8574 mmol) of benzamidine. The reaction mixture was allowed to stirunder nitrogen for 2 hours at ambient temperature, then was diluted withethyl acetate and washed with portions of H₂ O and brine. The organicphase was dried over magnesium sulfate, filtered and concentrated undervacuum to yield crude product. Purification by flash columnchromatography (elution with 60% then 80% ethyl acetate/hexanes ) gave0.1032 g (10%) of the title compound as a pale yellow solid.

¹ H NMR (DMSO) δ 9.05 (m, N-H, 2H), 7.98-7.48 (m, aromatic-H, 5H), 4.48(m, 42C-H, 1H).

MS (-) FAB m/z: 1059 (M⁻), 590 (Southern Fragment), 467 (NorthernFragment).

Results obtained in standard pharmacological test procedures:

LAF IC₅₀ : 2.05 nM

LAF ratio: 0.47

Skin graft survival: 9.8±0.8

Percent change in adjuvant arthritis versus control: -91%

EXAMPLE 3 Rapamycin 42-ester with (imino-pyridin-2-yl)methyl)carbamicacid

To 0.585 g (3.7 mmol) of 2-amidinopyridine hydrochloride was added oneequivalent of 0.1M sodium hydroxide/methanol after which the solvent wasremoved in vacuo. To the solution of the free base in 20 ml of DMF wasadded 4.0 g (3.7 mmol) of 42-O-(4-Nitro-phenoxycarbonyl)rapamycin. Thereaction mixture was allowed to stir under nitrogen for 6 hours atambient temperature, then was diluted with ethyl acetate and washed withportions of H₂ O and brine. The organic phase was dried over magnesiumsulfate, filtered and concentrated under vacuum to yield crude product.Purification by flash column chromatography (elution with 1:1 ethylacetate: hexanes, then 100% ethyl acetate) gave 0.47 g (12%) of thetitle compound as an off-white solid.

¹ H NMR (DMSO) δ 9.05 (m, N-H, 2H), 8.68-8.72 (m, Ar-H, 1H), 8.28-8.24(m, Ar-H, 1H), 8.0-7.94 (m, Ar-H, 1H), 7.66-7.62 (m, Ar-H, 1H), 4.4 (m,42C-H, 1H).

MS (-) FAB m/z: 1060 (M⁻), 590 (Southern Fragment), 468 (NorthernFragment).

Results obtained in standard pharmacological rest procedures:

LAF IC₅₀ : 0.60 and 0.55 nM

LAF ratio: 1.50 and 0.78

Skin graft survival: 11.7±1.0

What is claimed is:
 1. A method of treating transplantation rejectiongraft vs. host disease in a mammal in need thereof, which comprisesadministering to said mammal an antirejection effective amount of acompound of the structure ##STR5## wherein R and R¹ are each,independently, hydrogen, or ##STR6## R² and R³ are each, independently,hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, --CO₂ R⁵, --COR⁵, --CN, --NO₂, --SO₂ R⁵,--SO₃ R⁵, --OR⁵, --SR⁵, or Ar;R⁴ is hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, --CF₃, --NR⁵R⁶, --CO₂ R⁵, --COR⁵, CONR⁵ R⁶, --NO₂, halogen, --OR⁵, --SR⁵, --CN,--SO₂ R⁵, --SO₃ R⁵, --SO₂ NR⁵ R⁶, or Ar; R⁵ and R⁶ are each,independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7carbon atoms, alkynyl of 2-7 carbon atoms, or Ar; Ar is phenyl,naphthyl, or hetaryl, wherein the foregoing may be optionally mono-,di-, or tri-substituted with a group selected from alkyl of 1-6 carbonatoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo,hydroxy, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl,trifluoromethoxy, amino, dialkylamino of 1-6 carbon atoms per alkylgroup, dialkylaminoalkyl of 3-12 carbon atoms, hydroxyalkyl of 1-6carbon atoms, alkoxyalkyl of 2-12 carbon atoms, alkylthio of 1-6 carbonatoms, --SO₃ H, and --CO₂ H; hetaryl is a heterocyclic radical selectedfrom the group consisting of furanyl, thiophenyl, pyrrolyl, pyrazolyl,imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2-dithiolyl,1,3-dithiolyl, 1,2,3-oxathiolyl, isoxazolyl, oxazolyl, thiazolyl,isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,3,4-oxatriazolyl, 1,2,3,5-oxatriazolyl, 1,2,3-dioxazolyl,1,2,4-dioxazolyl, 1,3,2-dioxazolyl, 1,3,4-dioxazolyl,1,2,5-oxathiazolyl, 1,3-oxathiolyl, 1,2-pyranyl, 1,4-pyranyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1,2,4-triazinyl,1,2,3-triazinyl, 1,2,4-oxazinyl, 1,3,2-oxazinyl, 1,2,6-oxazinyl,1,4-oxazinyl, isoxazinyl, 1,2,5-oxathiazinyl, 1,4-oxazinyl,o-isoxazinyl, p-isoxazinyl, 1,2,5-oxathiazinyl, 1,2,6-oxathiazinyl,1,3,5,2-oxadiazinyl, azepinyl, oxepinyl, thiepinyl, 1,2,4-diazepinyl,benzofuranyl, isobenzofuranyl, thionaphthenyl, indolyl, indolenyl,2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, benzpyrazolyl,benzisoxazolyl, benzoxazolyl, anthranilyl, 1,2-benzopyranyl, quinolinyl,isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl,pyrido[3,4-b]pyridinyl, pyrido[4,3-b]pyridinyl, pyrido[2,3-b]pyridinyl,1,3,2-benzozazinyl, 1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl,3,1,4-benzoxazinyl, 1,2-benzisoxazinyl, 1,4-benzisoxazinyl, carbazolyl,and purinyl; with the proviso that R and R¹ are both not hydrogen, or apharmaceutically acceptable salt thereof.